HPTLC-FLD-SERS For Rapid Quantitative Screening And Molecular Conformation Of Histamine Residues In Fish

Histamine was the most toxic biogenic amines.Its content was commonly used as an important indicator to evaluate the degree of freshness and corruption of food.Histamine widely existed in all kinds of food and organisms,especially in aquatic products,so researching a rapid method for screening histamine residues in food was of great significance for food safety.This work presented a highly selective surface enhanced Raman spectroscopy(SERS)method mediated by high-performance thin layer chromatography(HPTLC)and fluorescent densitometry(FLD)for the identification and quantitation of histamine.And the role of NaCl electrolyte in the silver colloid system was analyzed.Under optimized conditions,this method was used to identify histamine in different fish samples.A method based on HPTLC-FLD-SERS for rapid quantitative screening and molecular conformation of histamine residues in food was established.Following separation and derivatization with fluram,plates were assayed with SERS,jointly using silver nanoparticle and NaCl.Through investigation,the factors which could affect the sensitivity of HPTLC-FLD-SERS were optimized,including the method of derivatization,the scanning condition of fluorescent densitometry,the type of the TLC plates,the preparation method of silver nanoparticles and the excitation wavelength of SERS,etc.The result showed the concentration of histamine in the range of 50~600 ng/zone had a good linear correlation in HPTLC plates,and R~2 was 0.999.LOD of histamine was 16.9 mg/kg.The selective enhancement effects of different electrolyte on histamine target were compared and analyzed.NaCl as a"noise-tuner"showed the strongest effect of selective enhancement.2 mol/L NaCl as the best added concentration dramatically suppressed the masking effect caused by excessive fluram throughout the plate and offered intensive SERS fingerprints of histamine with 10~4 enhancement.More importantly,the synergic effects of the anion-cation combination of a given electrolyte on the selectivity of SERS detection were observed for the first time,which was contrary to conventional views.In addition,selective enhancement mechanism of NaCl which combined with the SERS peaks of histamine was analyzed.The surface layer of silver colloid could be modified by NaCl,and SERS was changed to act selectively on different compounds by balancing the affinity between target particles and silver particles.Optimized HPTLC-FLD-SERS method was applied to the rapid screening of histamine in different fish samples.Methanol:acetone:ammonia(25%)=3:7:0.5(v/v/v)were used as the developing agent of four kinds of fish samples,which successfully separated the interfering impurities.Histamine was not detected in fresh ribbonfish and fresh tuna,but it was detected in frozen ribbonfish and frozen tuna at the concentration of 53.8 mg/kg and 65.9mg/kg,respectively.Relative standard deviation of frozen ribbonfish and frozen tuna were8.3%and 8.8%.The spiking recovery rate of four fish samples was 80.0%~105.2%.Moreover,the HPLC-UV method of detecting histamine which was recommended by European Union was used to verify the results of the fish samples tested by HPTLC-FLD-SERS,which confirmed the applicability and accuracy of the HPTLC-FLD-SERS method.